UP-02.099 Expression of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) in Prostate Cancer

UNMODERATED POSTER SESSIONS

1/Redox Factor-1 in Castrationresistant Prostate Cancer Cells Sul C, Song K, Na Y, Lim J, Shin J, Hwang E, Oh T Chungnam National University Hospital, Daejon, South Korea Introduction and Objective: Advanced and castration-resistant prostate cancer has long been considered as a chemo-resistant disease. The objectives of this study are to characterize apurinic/apyrimidinic endonuclease 1/redox Factor-1 (Ape1/Ref-1) expression levels in human prostate cancer cell lines and evaluate the effect of siRNA targeting the DNA repair and redox signaling protein Ape1/Ref-1. Materials and Methods: Two castration resistant prostate cancer cell lines, Du 145 and PC-3 cells were used for this study. Ape1/Ref-1 was transfected by siRNA and identified by real time PCR, western blot and immunofluorescent staining using confocal laser scan microscopy. By the RT-PCR and confocal microscopy, expression of Ape1/Ref-1 was detected according to concentration of docetaxel at 1, 3, 5 day. Expression of Ape1/Ref-1, caspase-3 according to concentration of docetaxel was detected between Ape1/Ref-1 siRNA (⫺) and Ape1/Ref-1 siRNA (⫹) at 3 day. Cell viability according to concentration of docetaxel was analyzed by MTT assay. Results: The expression of Ape1/Ref-1 after siRNA transfection in Du 145 and PC-3 cells was 20-30%. Expression of Ape1/Ref-1 in Du 145 and PC-3 cells according to concentration of docetaxel was increased at 3 day. Ape1/ref-1 siRNA transfection chemosensitized Du 145 and PC-3 cell to docetaxel and apoptotic rates in Du 145 and PC-3 cells were significantly increased when Ape1/Ref-1 was combined with docetaxel. Conclusion: The present findings indicate that Ape1/Ref-1 may play significant roles in the acquisition of chemo-resistant phenotype in castration resistant prostate cancer cells and targeted knockdown of Ape1/Ref-1 may enhance the effects of cytotoxic chemotherapy in docetaxel resistant cells.

UP-02.098 The Novel AKT-Inhibitor AZD5363 Abrogates Androgen-Receptor Signaling and Delays HormoneSensitive and Castration-Resistant Prostate Cancer Progression in vivo Thomas C1, Lamoureux F1, Crafter C2, Davies B2, Zoubeidi A1, Gleave M1 1 The Vancouver Prostate Centre, University of British Columbia,

Vancouver, Canada, 2AstraZeneca, Alderley Park, Macclesfield, UK Introduction and Objective: The PI3K/ Akt pathway plays a major role in prostate cancer (PCa) progression and therefore is an attractive pharmacological target. In this study, we assessed the antitumoral effects of the novel Akt-pathway inhibitor, AZD5363, in vitro and in vivo in PCa. We hypothesize that this novel drug will not only inhibit PI3K/Akt downstream effectors but also affect the androgen-receptor (AR) signaling. Methods: LNCaP and C4-2 cells were treated with AZD5363 and submitted to western blot and cell viability assay. AZD5363 was tested for its ability to abrogate AR transcriptional activity. In vivo, we used the hormone-sensitive and castration-resistant (CRPC) LNCaP xenograft model. Mice were randomly treated with AZD5363 or control by oral gavage immediately (hormone-sensitive) or delayed (CRPC) after castration. Tumor volume and serum PSA were evaluated consistently. Survival analysis, target gene protein expression and immunohistochemistry were evaluated for each group and model. Results: Here we report that AZD5363 inhibits cell growth in a dose-dependent manner in both LNCaP and C4-2 cells. Moreover, it induces cell apoptosis as measured with PARP cleavage expression, caspase 3 activity, and increases the sub-G1 population. Interestingly, AZD5363 significantly abrogates androgen-induced PSA transactivation and down-regulates PSA protein expression at concentrations ⬎1␮M. These biologic events are accompanied by inhibition of downstream PI3K/ Akt signaling. Most important, targeting the Akt-pathway with AZD5363 in vivo significantly reduces tumor growth rate by 6.4-fold in the hormone-sensitive model and by 6.2-fold in the CRPC model compared to each control. Moreover, serum PSA velocity is reduced by 4.3-fold in the hormone-sensitive model and by 5.0-fold in the CRPC model compared to each control. Consistently, AZD5363 treatment significantly improves biochemical- and tumor progression-free survival. Conclusions: This study reports as a preclinical proof-of-principle that targeting PI3K/Akt pathway with AZD5363 inhibits hormone-sensitive and castration-resistant PCa progression in vitro and in vivo, using additionally the AR-axis as a pharmacodynamic tool.This work was supported by AstraZeneca, the Deutsche Forschungsgemeinschaft (German Research Foundation; Grant number: TH 1482/2-

UROLOGY 78 (Supplement 3A), September 2011

1), the Canadian Institutes of Health Research and l’Association pour la Recherche sur le Cancer (France).

UP-02.099 Expression of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) in Prostate Cancer Yasuda K, Morii A, Watanabe A, Fujiuchi Y, Komiya A, Fuse H Dept. of Urology, University of Toyama, Graduate School of Medicine and Pharmaceutical Sciences for Research, Toyama, Japan Objectives: Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is Kunitz-type serine protease inhibitor for hepatocyte growth factor activator and matriptase. We have demonstrated that serum HAI-1 levels were elevated in patients with advanced-stage prostate cancer. We investigated whether the expression of HAI-1 was associated with the progression of prostate cancer. Materials and Methods: We evaluated the expression of HAI-1 by immunohistochemistry (IHC) in 51 patients who were negative for prostate biopsy and 75 patients with prostate cancer. The relationship between HAI-1 expression and several clinicopathological factors and serum HAI-1 levels was analyzed. Furthermore, we evaluated expression of HAI-1 in 24 castration resistance prostate cancer (CRPC) patients by IHC. Progression free survival was analyzed according to HAI-1 expression. Results: The mean score for HAI-1 in patients with negative for biopsy and prostate cancer patients were 1.75⫾0.74 and 2.24⫾0.87, respectively. Expression of HAI-1 in patients with prostate cancer was significantly higher than those with negative for biopsy patients (P⫽0.0002). There were no significant differences in HAI-1 expression with respect to clinical stage or tumor grade. But CRPC patients had significantly lower HAI-1 expression than pretreatment metastatic prostate cancer patients (P⫽0.046). In addition, no relationship was found between the HAI-1 score of tumor tissues and serum HAI-1 level. PSA progression free survival rate was lower in prostate cancer patients with negative HAI-1 expression in pretreatment biopsy specimens than those with positive HAI-1 expression (P⫽0.031). Conclusions: It was suggested that HAI-1 expression by IHC was related to tumor development and androgen-independent progression in prostate cancer. HAI-1 expression was lower in biopsy specimens

S293

UNMODERATED POSTER SESSIONS

of CRPC patients. However, serum HAI-1 showed higher values in CRPC patients. There is a possibility that release of membrane-form HAI-1 was enhanced in prostate cancer epithelial cell surface.

UP-02.100 Antitumor Effect of Sodium Metaarsenite on the Prostate Cancer Cell and Its Mechanism Lim JH, Yoo DS, You D, Kim Y, Jeong IG, Hong B, Hong JH, Ahn H, Ahn TY, Kim CS Dept. of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea Introduction and Objective: We investigated the effects of sodium metaarsenite on the cell growth inhibition and cell death in human prostate cancer cell lines. Additionally, we analyzed the role of autophagy as a mechanism by which sodium metaarsenite treatment induced cell death. Materials and Methods: Human prostate cancer cells including PC3, DU145, LNCaP and sodium metaarsenite (NaAsO2) were used as materials. Cell viability was assessed using the cell proliferation reagent Alamar blue (Alamar blue assay) and expression of protein was analyzed by Western blot. Apoptosis and autophagy were inhibited by adding Z-VAD-FMK or 3-MA, respectively. Results: Incubation of PC3, DU145 and LNCaP prostate cancer cells with 0.01-100 ␮M sodium metaarsenite for 48 hours reduced cell viability in a dose-dependent manner. LNCaP cells were more sensitive than PC3 or DU145. Sodium metaarsenitetreated cells showed both cells positive for annexin V staining only and positive for annexin V and DNA staining. These results indicate that cell death by sodium metaarsenite was induced by apoptosis and necrosis. Exposure of prostate cancer cells to sodium metaarsenite resulted in enhanced cleavage of PARP and activation of caspases-3 in time and dose-dependent manners. After exposure of prostate cancer cells to sodium metaarsenite, expression of LC3-II increased relative to controls, which was accompanied by a decrease of LC3-I expression. The amount of LC3-II demonstrated a cumulative increase in time and dose-dependent manners. Expression of LC3-II decreased with addition of 3-MA 2 mM in all prostate cancer cells. Taken together, all these observations support the hypothesis that autophagy was induced by sodium metaarsenite in prostate cancer cells. Conclusions: Our results indicate that

S294

exposure to sodium metaarsenite activates both autophagic cell death and apoptosis in androgen-dependent and independent prostate cancer cells. These findings suggest that sodium metaarsenite should be further investigated as a novel chemotherapeutic agent for the medical treatment of prostate cancer.

UP-02.101 BK Polyomavirus Infection in Prostate Tissue Derived from Patients with Prostate Adenocarcinoma Zhong S1, Suzuki M2, Shen Z1, Li T1, Wang X1, Yogo Y2, Homma Y2 Depts. of Urology, 1Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, 2Graduate School of Medicine, University of Tokyo, Tokyo, Japan Introduction and Objective: BK polyomavirus (BKV) has been proposed to be involved in the etiology of prostate cancer. As persistent infection with BKV in prostate tissue may be a prerequisite for this hypothesis, we examined whether BKV DNA sequences are prevalent in tissues of prostate adenocarcinoma, and investigate the potential role of BKV in prostate tumorigenesis. Materials and Methods: There were 32 consecutive patients who underwent radical prostatectomy because of prostate adenocarcinoma were included in the study. Tissue specimens were obtained from two regions of the prostate: one proximal and the other distal from the region used for pathological examination. Total DNA was extracted and subjected to single or nested polymerase chain reaction (PCR) to detect sequences within the large T antigen (LTag) gene, the VP1 gene and the transcriptional control region (TCR). In parallel, we determined not only the sensitivity of our PCR but also the amount of cellular DNA used for each PCR using the ß-globin gene as the marker. Results: BKV DNA was detected in distal regions is in 6 of the 32 cases (18.8%). In no case BKV DNA was detected from proximal regions. However, viral sequences were detected in neither the distal nor proximal regions from the other 26 patients (81.2%).All PCR targets were amplified in four cases, only the LTag was amplified in one case, and the LTag and TCR were amplified in one case. We sequenced all detected BKV DNA fragments. Conclusions: We hypothesized that persistent BKV infection would generally occur in the prostate tumor tissue, and that during persistence, virus-cell interaction

would be modulated in frequent infected cells, so that BKV replication is repressed, with LTag and STag expressed continuously, leading to the development of prostate cancer. The present data suggests that BKV infection plays a relevant role in the development and progression of human prostate adenocarcinoma, and possibly through the function of the LTag and TCR.

UP-02.102 Lithuania Prostate Cancer Early Diagnosis Program 2006-2010: Trends in Services Adomaitis R, Jankevicius F Clinic of Gastroenterology, Nephrourology and Surgery, Vilnius University, Vilnius, Lithuania Introduction and Objectives: To examine participation of men in the Lithuanian Prostate Cancer Early Diagnosis Program (LPCEDP) with regard to their age, as age is known to be an important indicator predicting the likelihood of men to suffer from prostate cancer and the opportunity to benefit from early detection and treatment. Materials and Methods: Data for the years 2006-2010 was analyzed: males 50-75 years old, visits to General practitioners (GPs) and the use of LPCEDP services. Odds ratios for two subgroups of men were calculated in view of Lithuanian estimated 70-year-olds life expectancy (10.9 years): born in 1941 and younger (“50 ⫹”), and born in 1942 and older (“70 ⫹”). In calculations subgroup “70 ⫹” was considered to be a reference. Results: In 2006-2010, 522717 men were identified who were eligible for LPCEDP PSA test. The “50 ⫹” subgroup accounted for 76.6% of LPCEDP population. Within five years 20.3% of men have never been to GP, and have no possibility to participate in LPCEDP. There was 84.4% of GP non-attendees who fall into the “50 ⫹” subgroup. Among men alive at the end of 2010 LPCEDP, penetration reached 49.8%. For man from the “50 ⫹” subgroup to check PSA level in LPCEDP estimated odds ratio was OR 0.787 (95% CI 0.775 to 0.798). For man from “70 ⫹” subgroup to be checked more than once estimated OR 1.209 (95% CI 1.191 to 1.228), and to be checked three or more times OR 1.386 (95%CI 1.355 to 1.417). Conclusions: it was found that in “70 ⫹” subgroup males significantly more often use the PSA check in LPCEDP. Such trends in LPCEDP services in future may have a significant impact on possibility to

UROLOGY 78 (Supplement 3A), September 2011